探討益生菌改善慢性代謝疾病之機轉

Studies on the mechanisms of Lactobacillus on ameliorating the complications in chronic metabolic diseases

謝豐欽(Feng-Chin Hsieh)

指導教授 ： 吳慶軒

高雄醫學大學/健康科學院/醫學檢驗生物技術學系博士班/博士(2017年)

摘要

根據統計，罹患第二型糖尿病的人占所有糖尿病患人口高達80%，為最常見之糖尿病類型。目前已經有許多研究證實攝取足量之益生菌對人體的健康有許多的助益，包括糖尿病發生之預防與症狀之改善。已經有許多研究報告證明給第一型糖尿病實驗動物餵食乳酸菌可以降低血糖以及其併發症。此外，在我們先前的研究已經證明 Lactobacillus reuteri GMNL-263 (Lr263) 可以改善 Streptozocin (STZ) 所誘導的第一型糖尿病老鼠的血糖值以及高血糖所誘發的腎纖維化。雖然已經有很多研究均證實乳酸菌可以治療或預防第一型糖尿病，然而乳酸菌是否可以改善第二型糖尿病及預防併發症之形成仍未知。因此，在本研究中我們設計了兩個實驗來研究乳酸菌是否可以改善代謝症候群以及其可能的作用機轉。
在第一個實驗中，我們使用雄性 Sprague–Dawley 大鼠同時餵食高果醣飼料以及 Lr263乳酸菌，經過14周後分別測量大鼠的糞便菌相以及口服葡萄糖耐受性。接著，我們偵測餵食 Lr263乳酸菌的大鼠血清中的糖尿病相關生化指標、肝功能指標以及血脂值濃度變化。除此之外，我們也有測量脂肪組織中的發炎指標以及胰島素訊息傳遞基因的變化。而為了探討 Lr263乳酸菌對於改善脂肪肝的效果，我們分別分析餵食 Lr263大鼠肝臟中的脂肪濃度、抗氧化酵素活性以及肝臟中脂肪合成相關基因的變化。最後，我們將肝臟組織病理切片用 hematoxylin-eosin染色法來觀察Lr263改善肝臟中脂肪堆積的效果。從實驗結果可以發現餵食Lr263乳酸菌的糖尿病大鼠可以顯著的改善其血清中的糖尿病相關指標、肝功能指標以及血脂值濃度。而餵食 Lr263也可以顯著的改善糖尿病大鼠糞便中的菌相，使糞便中的比菲德氏菌以及乳酸菌菌數增加，並同時減少糞便中的產氣夾膜菌的菌數。除此之外，我們也發現餵食 Lr263可以顯著的增加肝臟中的抗氧化酵素活性以及減少脂肪組織中的發炎指標。讓我們最感到訝異的是餵食Lr263的糖尿病大鼠，其體內的胰島素訊息傳遞相關基因以及脂肪合成相關基因也都可以顯著的獲得改善。
第二個實驗之研究目的是研究並比較 Lactobacillus reuteri GMNL-263 (Lr263)熱殺死菌以及活菌改善高脂飼料誘發肥胖大鼠的胰島素阻抗以及其他相關併發症。用高脂飼料餵食雄性 Sprague-Dawley(SD)大鼠，同時餵食 Lr263熱殺死菌或活菌。經過12週後我們發現餵食Lr263熱殺死菌或活菌的大鼠體重增加，血糖，胰島素濃度以及血清以及肝臟中的脂肪都顯著的比單純餵食高脂飼料組大鼠來的低。此外，餵食 Lr263熱殺死菌或活菌也可以改善因高脂所引起的致病菌菌數增加以及益生菌菌數減少的情況。Lr263熱殺死菌或活菌可以治療高脂飼料所引起的腸道屏蔽損傷。我們也發現餵食高脂飼料大鼠的脂肪以及肝臟組織中促發炎相關基因的表現顯著的提高。然而,若同時餵食 Lr263熱殺死菌或活菌的大鼠脂肪以及肝臟組織中促發炎相關基因的表現則會被抑制下來。值得注意的是我們觀察到在餵食高脂飼料後大鼠的脂肪組織巨噬細胞浸潤的現象會顯著增加。但是餵食 Lr263熱殺死菌或活菌則可以顯著的改善脂肪組織巨噬細胞浸潤的現象。單純餵食高脂飼料大鼠脂肪以及肝臟組織的胰島素敏感性相關基因表現都被顯著的抑制，然而餵食 Lr263熱殺死菌或活菌的大鼠則可以改善胰島素敏感相關基因的表現。除此之外，攝食 Lr263熱殺死菌或活菌可以改善因為餵食高脂飼料而被刺激增加表現肝臟中的脂質生成相關基因。通過觀察肝臟組織油紅 O染色(oil red o stain)我們發現餵食Lr263熱殺死菌或活菌可以改善大鼠脂肪肝的狀況。經由本篇研究結果我們發現 Lr263熱殺死菌或活菌可以藉由改善胰島素阻抗以及脂肪肝行程而達到降低因肥胖所引起的代謝異常且其效果非常相似。
由本實驗結果可以證明食用益生菌Lr263可以有效的改善胰島素阻抗以及脂肪肝。而這一結果可以提供人們一個更安全以及有效的方法來改善代謝症候群以及其併發症。

關鍵字

乳酸菌 ； 代謝症候群 ； 糖尿病 ； 肥胖 ； Lr263

並列摘要

Type 2 diabetes mellitus (DM), characterized by peripheral insulin resistance, is the most common form of diabetes comprising 80 % of all diabetic population. Probiotics are living microorganisms when taken in adequate amount may provide benefits to the host including modulation of DM development. Several lines of evidences have demonstrated the efficacy of Lactobacillus on reducing blood glucose and preventing the development of diabetic complications in type 1 diabetic animal models. Our previous studies also showed that live Lactobacillus reuteri GMNL-263 (Lr263), a common probiotic strain which is commercially available as a healthy food in Taiwan, protected streptozotocin-induced rats (animal model of type 1 diabetes) from development of hyperglycemia-induced renal fibrosis. However, the effects of Lactobacillus on metabolic syndrome and its related complications have not been clarified. Therefore, our study aimed to elucidate this critical issue by performing two animal experiments and explored the possible mechanisms involved.
In the first animal experiment, male Sprague–Dawley rats were fed a high-fructose diet with or without Lr263 administration for 14 weeks. The composition of fecal microbiota, oral glucose tolerance, glycated haemoglobin, insulin, leptin, C-peptide, and incretins were measured. The markers of liver injury, serum and hepatic lipids profile, activity of hepatic antioxidant enzyme, and proinflammatory cytokines in adipose tissue were investigated. Additionally, the expression of hepatic lipogenic genes and insulin signaling related genes in adipose tissue were also studied. Liver sections were examined for hepatic steatosis using hematoxylin-eosin staining. The result showed that the levels of serum glucose, insulin, leptin, C-peptide, glycated hemoglobin, GLP-1, liver injury markers, lipid profile in serum and liver were significantly increased in high-fructose-fed rats. However, after Lr263 administration, the elevation of these parameters was significantly suppressed. Feeding of Lr263 reversed the decreased number of Bifidobacterium species and Lactobacillus species and increased number of clostridium species induced by high fructose treatment. The decreased activities of hepatic antioxidant enzymes in HFD rats were dramatically reversed by Lr263 treatment. Concentrations of IL-6 and TNF-α in adipose tissue which were elevated in high fructose treatment were markedly decreased after Lr263 feeding. Decreased levels of PPAR-γ and GLUT4 mRNA after high fructose treatment were significantly enhanced by Lr263 administration. Lr263 consumption normalized the increased lipogenic gene (Srebp-1c, FAS, and Elvol6) expressions stimulated by high fructose. Administration of Lr263 significantly ameliorated hepatic steatosis observed in high fructose treated rats.
The objective of second study was to investigate and compare the effects of heat-killed (HK) and live Lactobacillus reuteri GMNL-263 (Lr263) on insulin resistance and its related complications in high-fat diet (HFD)-induced rats. Male Sprague-Dawley rats were fed with a HFD with either HK or live Lr263 for 12 weeks. The increase in weight gain, serum glucose, insulin, and lipid profile in serum and liver observed in HFD group were significantly reduced after HK or live Lr263 administration. Feeding of HK or live Lr263 reversed the decreased number of probiotic bacteria and increased number of pathogenic bacteria induced by high-fat treatment. The decreased intestinal barrier in HFD group was markedly reversed by HK or live Lr263 treatments. The elevations of pro-inflammatory associated genes expressions in both adipose and hepatic tissues by high-fat administration were markedly decreased by HK or live Lr263 treatments. The increased macrophage infiltration noticed in adipose tissue after high-fat treatment was effectively suppressed by HK or live Lr263 consumption. The insulin resistance associated gene expressions in both adipose and hepatic tissues which were downregulated in HFD group were markedly enhanced after HK or live Lr263 feeding. HK or live Lr263 consumption significantly decreased hepatic lipogenic gene expressions stimulated by high-fat treatment. Administration of HK or live Lr263 significantly reduced hepatic oil red O staining and ameliorated hepatic steatosis observed in high-fat treated rats. Our data suggested that similar to live Lr263, HK Lr263 exerted significant effectiveness on attenuating obesity-induced metabolic abnormalities by reducing insulin resistance and hepatic steatosis formation.
Our study provided evidences clarifying the effectiveness of Lr263 on reducing insulin resistance as well as hepatic steatosis and suggested that Lr263 may be a promising healthy agent in improving metabolic syndrome.

並列關鍵字

Lactobacillus ； Metabolism syndrome ； Diabetes ； Obesity ； Lr263

參考文獻

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